This invention relates to rubber polymerases in substantially pure form and methods for their production and use in the in vitro synthesis of natural rubber, particularly high molecular weight natural rubber.
Natural rubber is found in thousands of plants from various families. Several fungi have also been reported to synthesize natural rubber. Most plants and all of the fungi contain very little rubber, and the molecular weight of the rubber is too low for the rubber to be of commercial value.
The two sources of natural rubber that have been used commercially are Hevea brasiliensis and Parthenium argentatum (guayule). H. brasiliensis is the dominant source of natural rubber. Guayule was a commercial source of natural rubber in the first part of this century, providing 10% of the world supply in 1910. During World War II, when the United States' supply of natural rubber from Southeast Asia was cut off, a large-scale planting of guayule was conducted by the U.S. Forest Service as part of the Emergency Rubber Project. When the war was over, the low price and high availability of H. brasiliensis rubber made guayule an unattractive source of natural rubber. The dominance of H. brasiliensis continues today. However, recent economic and political trends encourage the development of alternative sources of high molecular weight natural rubber. One of these sources is in vitro synthesis.
The pathway of rubber biosynthesis in H. brasiliensis is well established and has been reviewed by Archer and Audley, Advances in Enzymology, 29: 221-257, 1967 and Lynen, J. Rubber Res. Inst. Malaya, 21(4): 389-406, 1969. The final step in the biosynthesis of rubber is catalyzed by the enzyme rubber polymerase, which is found in the latex. This enzyme catalyzes the polymerization of isopentenyl pyrohosphate (IPP) onto an allylic pyrophosphate to make rubber with the production of inorganic pyrophosphate. The enzyme has also been called rubber transferase or cis-1,4-polyisoprene pyrophosphate: isopentenyl pyrophosphate cis-1,4-polyisoprenyl transferase. It is a member of the group of enzymes called prenyltransferases, which polymerize IPP to different chain lengths and stereochemical configurations. The term "rubber polymerase" will be used herein to describe the enzyme.
In H. brasiliensis latex, the enzyme is mainly associated with rubber particles, but it is also found free in solution, Archer and Audley, supra. The synthesis of rubber takes place on the surface of the rubber particles, Lynen, supra. Even though studies of rubber polymerase have been carried out in a number of laboratories (Archer and Audley, supra; Lynen, supra; Archer and Cockbain, Methods in Enzymology, 15: 476-481, 1969), successful production of rubber polymerase from H. brasiliensis or any other source in substantially pure form has not been achieved prior to the present invention.
Rubber polymerase in substantially pure form will have several applications that will be very important for the rubber industry. The enzyme, either immobilized or free in solution, could be used to produce natural rubber in vitro. This would allow the production of the rubber without the use of H. brasiliensis latex and at locations near factories where the rubber is processed into finished articles, such as tires. In addition, natural rubber made in vitro may even be superior to natural rubber produced from H. brasiliensis latex because the conditions for making natural rubber in vitro could be controlled so as to produce natural rubber of higher purity or having other desirable chemical or physical properties. For example, the in vitro rubber would be more uniform and would also be substantially free of proteins and lipids that can interfere with certain applications of natural rubber.
Another important application would to prepare antibodies to the substantially pure rubber polymerase. Such antibodies could be used to develop a screening method for plants and other organisms to determine the amount of rubber polymerase in such organisms.
In still another important application, the purified enzyme could be sequenced and, using the sequence of the enzyme, the rubber polymerase gene or a probe for the gene could be synthesized. Once the gene for the enzyme was identified, the ability to produce natural rubber could be introduced into other organisms. Finally, once more is known about the sequence and structure of the enzyme, site directed mutagensis could be used to produce a rubber polymerase with altered catalytic properties. This would allow more control over the biosynthesis of natural rubber in vivo and in vitro.